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Abstract
GlgE is a bacterial maltosyltransferase that catalyzes the elongation of a cytosolic, branched α-glucan. In Mycobacterium tuberculosis (M. tb), inactivation of GlgE (Mtb GlgE) results in the rapid death of the organism due to a toxic accumulation of the maltosyl donor, maltose-1-phosphate (M1P), suggesting that GlgE is an intriguing target for inhibitor design. In this study, the crystal structures of the Mtb GlgE in a binary complex with maltose and a ternary complex with maltose and a maltosyl-acceptor molecule, maltohexaose, were solved to 3.3 Å and 4.0 Å, respectively. The maltohexaose structure reveals a dominant site for α-glucan binding. To obtain more detailed interactions between first generation, non-covalent inhibitors and GlgE, a variant Streptomyces coelicolor GlgEI (Sco GlgEI-V279S) was made to better emulate the Mtb GlgE M1P binding site. The structure of Sco GlgEI-V279S complexed with α-maltose-C-phosphonate (MCP), a non-hydrolyzable substrate analogue, was solved to 1.9 Å resolution, and the structure of Sco GlgEI-V279S complexed with 2,5-dideoxy-3-O-α-D-glucopyranosyl-2,5-imino-D-mannitol (DDGIM), an oxocarbenium mimic, was solved to 2.5 Å resolution. These structures detail important interactions that contribute to the inhibitory activity of these compounds, and provide information on future designs that may be exploited to improve upon these first generation GlgE inhibitors.
Highlights
The glgE pathway of M. tb is one of three α -glucan biosynthetic pathways encoded by the M. tb genome[6]
A variant of the Sco GlgEI that has an M1P binding site more representative of the Mtb GlgE site was co-crystallized with two different classes of GlgE inhibitors and the X-ray crystal structures were solved
The differences observed in the homodimer from the SAXS experiments may be attributed to the presence of a disulfide bridge that covalently links the monomers in the Mtb GlgE crystal structure, while this is absent in the Sco GlgEI homolog
Summary
The glgE pathway of M. tb is one of three α -glucan biosynthetic pathways encoded by the M. tb genome[6]. The increase of M1P concentration elicits an apparent stress response by the bacterium that stimulates the over expression of biosynthetic enzymes necessary for the production of trehalose and more M1P. This positive feedback loop and overproduction of M1P causes pleiotropic effects that cause rapid bacterial death[5]. This effect is novel in that killing is the result of an over production of a toxic metabolite rather than the absence of an important metabolite. A variant of the Sco GlgEI that has an M1P binding site more representative of the Mtb GlgE site was co-crystallized with two different classes of GlgE inhibitors and the X-ray crystal structures were solved
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